The present invention relates to a process for producing N-acetylneuraminic acid by using a microorganism having N-acetylneuraminic acid aldolase activity or N-acetylneuraminic acid synthetase activity.
It is known that N-acetylneuraminic acid can be produced by extraction, decomposition or by use of enzymes.
An example of a known method by extraction is extraction from a nest of sea swallows, etc. [Carbohydrate Research, 56, 423 (1977)].
An example of a known method by decomposition is decomposition of colominic acid, which is an N-acetylneuraminic acid polymer produced by Escherichia coli, etc. [J. Biochem., 82, 1425 (1977)].
Known methods utilizing enzymes include the following: methods using N-acetylneuraminic acid aldolase, pyruvic acid and N-acetylmannosamine [J. Am. Chem. Soc., 110, 6481 (1988); J. Am. Chem. Soc., 110, 7159 (1988)]; a method using N-acetylneuraminic acid aldolase, pyruvic acid and N-acetylglucosamine under alkaline conditions (U.S. Pat. No. 5,665,574); methods using N-acetylneuraminic acid aldolase, N-acetylglucosamine 2-epimerase, pyruvic acid and N-acetylglucosamine [Angew. Chem. Int. Ed. Eng., 30, 827 (1991); Carbohydrate Research, 306, 575 (1998)]; and methods using N-acetylneuraminic acid synthetase, phosphoenolpyruvic acid and N-acetylmannosamine [Japanese Published Unexamined Patent Application No. 4961/98; Glycobiology, 7, 697 (1997)].
The above methods for producing N-acetylneuraminic acid require complicated operations or expensive materials such as pyruvic acid and phosphoenolpyruvic acid, and an economical method for producing N-acetylneuraminic acid has not been established yet.
So far, there has been no report describing or suggesting that N-acetylneuraminic acid can be produced by utilizing a culture of a microorganism or a treated matter thereof.
As for the N-acetylneuraminic acid aldolase, those derived from animals and plants are known, and it is known that microorganisms belonging to the genus Escherichia have the activity of this enzyme. Also known is the presence of the gene encoding this enzyme, nanA, in an Escherichia coli strain [Nucleic Acids Res., 13, 8843 (1985)].
N-Acetylneuraminic acid synthetase is known to be present in microorganisms belonging to the genera Escherichia, Neisseria and Streptococcus, etc., and it is known that an Escherichia coli strain has the gene encoding this enzyme, neuB [J. Bacteriol., 177, 312 (1995)].
N-Acetylglucosamine 2-epimerase is known to be present in pigs and rats. The properties of the enzyme derived from pig have been investigated [Biochemistry, 17, 3363 (1970)] and the gene encoding the enzyme [J. Biol. Chem., 271, 16294 (1996)] has been obtained. So far, no microorganism having the activity of this enzyme is known.
As to the production of pyruvic acid, a process for producing pyruvic acid by using a mutant of Escherichia coli is known [Biosci. Biotech. Biochem., 58, 2164 (1994)].
As to the production of phosphoenolpyruvic acid, a process for producing phosphoenolpyruvic acid by using microorganisms of Saccharomyces, etc. is known (Japanese Published Unexamined Patent Application No. 197778/94).
An object of the present invention is to provide a process for economically producing N-acetylneuraminic acid without using expensive materials such as pyruvic acid and phosphoenolpyruvic acid. A further object of the present invention is to provide a process for producing N-acetylneuraminic acid without using expensive N-acetylmannosamine.